Methods of treatment

ABSTRACT

A method of inducing inhibition or loss of cell proliferation in solid tumors utilizing a Vitamin D 3  analog, as well as a method of inducing loss of cell proliferation in solid tumors utilizing trans retinoic acid and a Vitamin D 3  analog. 
     In another aspect, a method of inducing inhibition or loss of cell proliferation in solid tumors utilizing a Vitamin D 3  metabolite, as well as a method of inducing loss of cell proliferation in solid tumors utilizing trans retinoic acid and a Vitamin D 3  metabolite.

This is a division, of application Ser. No. 08/347,539, filed Nov. 30,1994, now U.S. Pat. No. 5,547,947, which is a Rule 60 Continuation ofSer. No. 08/029,744, filed Mar. 11, 1993, now abandoned.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention relates to a method of inducing inhibitionor loss of cell proliferation in solid tumors, particularly, breasttumors, which comprises administering to a host in need of suchtreatment an effective amount of a Vitamin D₃ analog. In another aspect,the invention relates to a method of inducing inhibition or loss of cellproliferation in solid tumors, particularly, breast tumors whichcomprises administering to a host in need of such treatment an effectiveamount of trans retinoic acid and a Vitamin D₃ analog. Preferably, theVitamin D₃ analog is selected from the group consisting of

1α,25-dihydroxy-26,27-hexafluorocholecalciferol;

1α,25-dihydroxy-22-ene-26,27-hexafluorocholecalciferol;

1α,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol;

1α,25-dihydroxy-23-yne-cholecalciferol;

1α,25-dihydroxy-16-ene-cholecalciferol;

1α,25-dihydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol;

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol; and

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol.

In yet another aspect, the invention relates to a method of inducinginhibition or loss of cell proliferation in solid tumors, particularly,breast tumors, which comprises administering to a host in need of suchtreatment an effective amount of a Vitamin D₃ metabolite, such as,1α,25-dihydroxycholecalciferol (hereinafter also referred to as "D₃ "),either alone or in combination with trans retinoic acid.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has now been found that a beneficial inhibition or lossof cell proliferation in solid tumors can be achieved utilizing aVitamin D₃ analog, especially one of those more particularly describedhereinafter, alone or in combination with all trans retinoic acid(hereinafter also referred to as "RA").

Accordingly, the invention comprises a method of achieving an inhibitionof or loss in cell proliferation in solid tumor in a host requiring suchtreatment by administering an effective amount of a Vitamin D₃ analogselected from the group consisting of

1α,25-dihydroxy-26,27-hexafluorocholecalciferol (hereinafter alsoreferred to as "HF");

1α,25-dihydroxy-22-ene-26,27-hexafluorocholecalciferol (hereinafter alsoreferred to as "22 HF");

1α,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol(hereinafter also referred to as "16-23HF");

1α,25-dihydroxy-23-yne-cholecalciferol (hereinafter also referred to as"23D");

1α,25-dihydroxy-16-ene-cholecalciferol (hereinafter also referred to as"16D");

1α,25-dihydroxy-16-ene-23 -yne-cholecalciferol (hereinafter alsoreferred to as "16-23D");

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol(hereinafter also referred to as "IF-16-23HF");

26,26,26,27,27,27-hexafluoro-1α,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol (hereinafter alsoreferred to as "19 nor");

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol; and

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol.

The invention also comprises a method of inducing inhibition or loss ofcell proliferation in solid tumors, particularly, breast tumors, whichcomprises administering to a host in need of such treatment an effectiveamount of a Vitamin D₃ metabolite, preferably,1α,25-dihydroxycholecalciferol, either alone or in combination withretinoic acid.

A preferred group of Vitamin D₃ analogs, utilized in the methods of theinvention, comprises the group:

1α,25-dihydroxy-26,27-hexafluorocholecalciferol;

1α,25-dihydroxy-22-ene-26,27-hexafluorocholecalciferol;

1α,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol;

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol;and

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol.

Preferred Vitamin D₃ analogs utilized in the methods of the inventionare 1α,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol;1α,25-dihydroxy-26,27-hexafluorocholecalciferol; and1α,25-dihydroxy-16-ene-23-yne-cholecalciferol.

A particularly preferred Vitamin D₃ analog, utilized in the methods ofthe invention, is1α,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol.

Processes for preparing 1α,25-dihydroxy-26,27-hexafluoro-cholecalciferolare set forth in U.S. Pat. No. 4,358,406, issued Nov. 9, 1982 which ishereby incorporated by reference.

Processes for preparing1α,25-dihydroxy-22-ene-26,27-hexafluoro-cholecalciferol are set forth inU.S. Pat. No. 4,613,594, issued Sep. 23, 1986 which is herebyincorporated by reference. Processes for preparing1α,25-dihydroxy-23-yne-cholecalciferol are set forth in U.S. Pat. No.4,804,502, issued Feb. 14, 1989 which is hereby incorporated byreference. Processes for preparing1α,25-dihydroxy-16-ene-cholecalciferol and1α,25-dihydroxy-16-ene-23-yne-cholecalciferol are set forth in U.S. Pat.No. 5,087,619, issued Feb. 11, 1992 which is hereby incorporated byreference. Processes for preparing all rans retinoic acid are set forthin U.S. Pat. No. 3,746,730, issued Jul. 17, 1973 which is herebyincorporated by reference. Processes for preparing

1α,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol;

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol;and

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol

are set forth in U.S. patent application Ser. No. 971,788, filed Nov. 5,1992, which is hereby incorporated by reference.

The methods of the invention can be utilized for the treatment of solidtumors, in particular, breast tumors, such as, those comprisingMDA-MB231 cells which are devoid of estrogen receptors, T-47D cellswhich possess estrogen receptors and the like. The inhibition or loss incell proliferation in solid tumors can be demonstrated utilizing theprocedures described herein below.

A colony assay can be used to evaluate inhibition or loss of solid tumorcell proliferation on tumor cells with the Vitamin D₃ analogs ormetabolites referred to herein, particularly,1α,25-dihydroxycholecalciferol;1α,25-dihydroxy-26,27-hexafluorocholecalciferol;1α,25-dihydroxy-22-ene-26,27-hexafluorocholecalciferol;1α,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol;1α,25-dihydroxy-16-ene-cholecalciferol; and1α,25-dihydroxy-16-ene-23-yne-cholecalciferol.

METHOD

Cells of the cell line utilized are trypsinized, counted and plated 250cells/5 ml minimal essential medium (MEM) containing 10% heatinactivated fetal calf serum. The test compound is added 48 hours afterplating for an exposure time of 120 hours. Thereafter, the medium isaspirated and replaced with drug-free medium, and 14-16 days later themedium is aspirated. The colonies formed are fixed and stained with 0.5%methylene blue in 50% methanol. Colonies which contain greater than 500cells are counted. The percent loss in colony formation is determined asfollows: ##EQU1##

The results are provided in Tables I-IV.

                  TABLE I    ______________________________________    EFFECT OF VITAMIN D ANALOGS ON COLONY FORMATION    BY HUMAN BREAST CARCINOMA CELLS (T-47-D)             Concentration                         Exposure time                                     % loss in    Drug     (μM)     (hr)        colony formation.sup.a    ______________________________________    D.sub.3  0.05        120         29*    D.sub.3  0.1         120         94*    16 D.sub.3             0.01        120         30.5 ± 6.4    16-23 D.sub.3             0.05        120         82.0 ± 11.3    23D.sub.3             0.1         120         72.0 ± 29.7    HF       0.01        120         83.5 ± 12.0    22HF     0.01        120         51.0 ± 45.2    16-23-D.sub.3 HF             0.01        120         100 ± 0    ______________________________________     .sup.a mean ± S.D. n = 2     *only one test was run at this concentration, accordingly no standard     deviation is provided.

                  TABLE II    ______________________________________    EFFECT OF VITAMIN D ANALOGS ON COLONY FORMATION    BY HUMAN BREAST CARCINOMA CELLS (MDA-MB231)             Concentration                         Exposure time                                     % loss in    Drug     (μM)     (hr)        colony formation.sup.a    ______________________________________    D.sub.3  0.1         120         25.0 ± 8.7    16 D.sub.3             0.01        120         42.0 ± 20.5    16-23 D.sub.3             0.05        120         30.0 ± 16.1    23D.sub.3             0.1         120         33.3 ± 14.9    HF       0.01        120         23.7 ± 19.8    22HF     0.01        120         29.0 ± 15.9    16-23-D.sub.3 HF             0.01        120         26.7 ± 17.3    ______________________________________     .sup.a mean ± S.E. n = 3

The synergistic compositions and methods of the invention can beutilized for the treatment of solid tumors, in particular, breasttumors. The inhibition or loss of solid tumor cell proliferation of themethod of the invention comprising administering a combination of transretinoic acid and Vitamin D₃ analogs or metabolites may be demonstratedutilizing the procedure described above. In order to determine if theinteractions between the drugs was synergistic the data on colonyformation were analyzed according to F. Valeriote and H. Lin,Synergistic Interaction of Anticancer Agents: A cellular perspective,Cancer Chemother. Rep. 59, 895 (1975). The results on percent loss incolony formation are presented in Tables III and IV below.

                  TABLE III    ______________________________________    EFFECT OF ALL-TRANS RETINOIC ACID (RA) AND 16-23D.sub.3    ON COLONY FORMATION BY HUMAN BREAST    CARCINOMA CELLS (MDA-MB-231)              Concentration                         Exposure time                                     % loss in    Drug      (μM)    (hr)        colony formation.sup.a    ______________________________________    RA        1.0        120         1.7 ± 0.9    16-23-D.sub.3              0.05       120         30.0 ± 16.1    RA + 16-23-D.sub.3              1.0 + 0.05 120         55.7 ± 10.6    ______________________________________     .sup.a mean ± S.E. n = 3

                  TABLE IV    ______________________________________    EFFECT OF ALL-TRANS RETINOIC ACID (RA) AND 16-23D.sub.3 HF    ON COLONY FORMATION BY HUMAN BREAST    CARCINOMA CELLS (MDA-MB-231)               Concentration                          Exposure time                                     % loss in    Drug       (μM)    (hr)       colony formation.sup.a    ______________________________________    RA         1.0        120        1.7 ± 0.9    16-23-D.sub.3 HF               0.01       120        26.7 ± 17.3    RA + 16-23-D.sub.3 HF               1.0 + 0.01 120        64.3 ± 18.7    ______________________________________     .sup.a mean ± S.E. n = 3

METHOD

Tetrazolium based MTT assay

T47D cells are seeded at densities of 4×10³ (cells/well) and 8-10³(cells/well) (day zero). Twenty four hours after (day one) seeding thetest compound is added to each well. On the second through seventh days,50 μl 3(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide isadded to each well. The plates are incubated at 37° C. for 2.5 hours.Then, the plates are centrifuged for 5 minutes at 800×g, supernantant isaspirated from the wells and 50 μl 100% ethanol is added to each well.The plates are then shaken for 15 minutes to solubilize formazan. A bluecolor developes in replicating cells. The optical density is measured inanautomatic plate reader at dual wavelength of 570/660 nm and comparedwith the optical density of a control. The IC₅₀ value reported is theconcentration of compound tested at which 50% of cell proliferation isinhibited as determined by the method of Reed and Muench, as describedin Reed, L. J., and H. Muench, A Simple Method of Estimating 50%endpoints, Am. J. Hyg. 27:493-497 (1938).

The results are as set forth in Table V below.

                  TABLE V    ______________________________________    IC.sub.50    Test Compound 4 × 10.sup.3 cells/well                              8 × 10.sup.3 cells/well    ______________________________________    16-23HF       1.3     μM     1.61  μM    1F-16-23HF    14.2    nM        22.3  nM    19 nor        9.0     nM        15.2  nM    D.sub.3       3.7     nM        8.9   nM    ______________________________________

The percent loss in colony formation is an index of the irreversibleloss of the proliferative potential of the tumor cells and is directlyproportional to the tumor kill potency of the Vitamin D₃ analog ormetabolite, alone or in combination.

The individual components of the method comprising administering transretinoic acid and a compound selected from the group consisting of

1α,25-dihydroxy-26,27-hexafluorocholecalciferol;

1α,25-dihydroxy-22-ene-26,27-hexafluorocholecalciferol;

1α,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol;

1α,25-dihydroxy-23-yne-cholecalciferol;

1α,25-dihydroxy-16-ene-cholecalciferol;

1α,25-dihydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-cholecalciferol;

26,26,26,27,27,27-hexafluoro-1α,25-dihydroxy-16-ene-23-yne-19-nor-cholecalciferol;

26,26,26,27,27,27-hexafluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol;and

26,26,26,27,27,27-hexafluoro-1α-fluoro-25-hydroxy-16-ene-23-yne-19-nor-cholecalciferol

may be administered either sequentially or simultaneously in separate orcombined pharmaceutical formulations.

The individual components of the method of comprising administeringtrans retinoic acid and 1α,25-dihydroxycholecalciferol may beadministered either sequentially or simultaneously in separate orcombined pharmaceutical formulations.

A composition of the invention may be administered by any of the modesby which the components may be administered, for example, orally,intravenously or the like. The dosage regiment may be regulatedaccording to the potency of individual compounds employed, the mode ofadministration, and the needs of the host mammal depending on factorssuch as the degree and the severity of the disease state and age andgeneral condition of the host mammal being treated.

In the method of the invention, trans retinoic acid and/or a Vitamin D₃analog or metabolite can be administered in unit dosage forms such astablets, capsules, pills, powders, granules, sterile parenteralsolutions or suspensions, suppositories, transdermal compositions,aerosols and the like. Such dosage forms are prepared according tostandard techniques in the art.

The dosages or therapeutically effective amounts utilized in the methodsof the invention may be varied depending upon the requirements of thehost, the severity of the condition being treated and the particularcompound being employed in the method. Determination of dose amount fora particular administration is within the skill of the art. Generally,treatment is initiated at lower dosages and increased as needed by smallincrements until the optimum effect is reached. Exemplary dosagesutilized in the methods of the invention are in the range of0.00025-0.01 mg Vitamin D₃ analog or metabolite per day, withappropriate monitoring of blood calcium levels and adjustment of thedosages as needed. For convenience, the total daily dosage may beadministered in divided doses.

Exemplary dosages of trans retinoic acid can be in the range of fromabout 10 to about 20 mg per day, preferably 14 mg per day. Generally thetrans retinoic acid is present in a ratio of from about 100 to about1000 parts to one part of the Vitamin D₃ analog or metabolite.

Oral dosage forms comprising compounds of formula I of the invention maybe incorporated in capsules, tablets and the like with one or morepharmaceutically acceptable carrier materials.

Illustrative of the pharmaceutically acceptable carrier materialsutilized in the pharmaceutical dosage forms are binders, such as gumtragacanth, acacia, corn starch or gelatin; an excipient, such as,dicalcium phosphate; disintegrating agents, such as, corn starch, potatostarch, algenic acid and the like; lubricants, such as, magnesiumstearate; sweetening agents, such as, sucrose, lactose or saccharin;flavoring agents, such as, peppermint, oil of wintergreen or cherry.Various other materials may be present as coating or to otherwise modifythe physical form of the dosage unit. For instance, tablets may becoated with shellac, sugar or both. A syrup or elixir may contain theactive compound, sucrose as a sweetening agent, a dye and a flavoringsuch as cherry or orange flavor.

Exemplary of formulations which may be utilized in the methods of theinvention are:

EXAMPLE 1

    ______________________________________    Capsule Formulation    Item    Ingredients       mg/capsule    ______________________________________    1       1α,25-dihydroxy-26,27-                               0.005   0.010            hexafluorocholecalciferol    2       Butylated Hydroxyanisole                               0.016   0.016    3       Butylated Hydroxytoluene                               0.016   0.016    4       Glycerin           16.000  16.000    5       PEG 400           143.963 143.958            TOTAL             160.000 160.000    ______________________________________

Manufacturing Procedure

Note

Perform all manufacturing steps under a nitrogen atmosphere and protectfrom light.

1. Warm the mixture of items 4 and 5 to 55° C.

2. Dissolve items 2 and 3 in the solution from Step 1.

3. Dissolve item 1 in the solution from Step 2.

EXAMPLE

    ______________________________________    Capsule Formulation    Item     Ingredients       mg/capsule    ______________________________________    1        1α,25-dihydroxy-22-ene-                               0.00025             26,27-hexafluorocholecalciferol    2        Polyethylene glycol 400                               200.00    3        Butylated Hydroxyanisole                               0.100    4        Ascorbyl palmitate                               1.00    ______________________________________

Manufacturing Procedure

Note

Perform all manufacturing steps under a nitrogen atmosphere and protectfrom light.

1. Dissolve items 1, 3 and 4 in item 2 and encapsulate.

EXAMPLE

    ______________________________________    Capsule Formulation    Item    Ingredients       mg/capsule    ______________________________________    1       1α,25-dihydroxy-26,27-                               0.005   0.010            hexafluoro-16-ene-23-yne            cholecalciferol    2       Butylated Hydroxyanisole                               0.016   0.016    3       Butylated Hydroxytoluene                               0.016   0.016    4       Neobee M-5        159.963 159.958            TOTAL             160.000 160.000    ______________________________________

Manufacturing Procedure

Note

Perform all manufacturing steps under a nitrogen atmosphere and protectfrom light.

1. Warm 10% of item 4 to 55° C.

2. Dissolve items 2 and 3 in the solution from Step 1.

3. Dissolve item 1in the solution from Step 2.

4. Add the remaining item 4 to the solution from Step 3 and mix well.

EXAMPLE

    ______________________________________    Capsule Formulation    Item    Ingredients       mg/capsule    ______________________________________    1       1α,25-dihydroxy-22-ene-23                               0.005   0.010            yne-cholecalciferol    2       Butylated Hydroxyanisole                               0.016   0.016    3       Butylated Hydroxytoluene                               0.016   0.016    4       Neobee M-5        159.963 159.958            TOTAL             160.000 160.000    ______________________________________

Manufacturing Procedure

Note

Perform all manufacturing steps under a nitrogen atmosphere and protectfrom light.

1. Warm 10% of item 4 to 55° C.

2. Dissolve items 2 and 3 in the solution from Step 1.

3. Dissolve item 1 in the solution from Step 2.

4. Add the remaining item 4 to the solution from Step 3 and mix well.

EXAMPLE

    ______________________________________    Capsule Formulation    Item    Ingredients       mg/capsule    ______________________________________    1       Trans Retinoic Acid                              10.00    2       1α,25-dihydroxy-16-ene-23                              0.01            yne-cholecalciferol    3       Purified Beewax   7.85    4       Hydrogenated Soyhean Oil                              7.85    5       Hydrogenated Vegetable Oil                              31.40    6       Butylated Hydroxyanisole                              0.11    7       Soybean Oil       107.28    8       Disodium Edetate  0.50            TOTAL             165.00    ______________________________________

Manufacturing Procedure

Note

Perform all manufacturing steps under a nitrogen atmosphere and protectfrom light.

1. Melt items 3, 4 and 5 by heating at 70° C., and mix well.

2. Dissolve item 6 in the mixture from Step 1.

3. Cool the mixture from Step 2 to 35°-40° C.

4. Add item 8 to the mixture from Step 3 and mix well.

5. Add item 7 to the mixture from Step 4, mix well and cool to roomtemperature.

6. Add items 1 and 2 to the mixture from Step 5 and disperse well untila uniform suspension is obtained.

EXAMPLE

    ______________________________________    Capsule Formation    Item      Ingredients     mg/capsule    ______________________________________    1         Trans Retinoic Acid                              10.000    2         1α,25-dihydroxy-26,27-                              0.00025              hexafluoro-16-ene-23-yne              cholecalciferol    3         Butylated Hydroxyanisole                              0.016    4         Butylated Hydroxytoluene                              0.016    5         Glycerin        16.000    6         PEG400          133.86775              TOTAL           160.000    ______________________________________

Manufacturing Procedure

Note

Perform all manufacturing steps under a nitrogen atmosphere and protectfrom light.

1. Warm the mixture of items 5 and 6 to 55° C.

2. Dissolve items 3 and 4 in the solution from Step 1.

3. Add items 1 and 2 to the solution from Step 2 and mix well.

EXAMPLE

    ______________________________________    Capsule Formulation    Item     Ingredients      mg/capsule    ______________________________________    1        Trans Retinoic Acid                              10.00    2        Purified Beewax  7.85    3        Hydrogenated Soybean Oil                              7.85    4        Hydrogenated Vegetable Oil                              31.40    5        Butylated Hydroxyanisole                              0.11    6        Soybean Oil      107.24    7        Disodium Edetate 0.50             TOTAL            165.00    ______________________________________

Manufacturing Procedure

Note

Perform all manufacturing steps under a nitrogen atmosphere and protectfrom light.

1. Melt items 2, 3 and 4 by heating at 70° C., and mix well.

2. Dissolve item 5 in the mixture from Step 1.

3. Cool the mixture from Step 2 to 35°-40° C.

4. Add item 7 to the mixture from Step 3 and mix well.

5. Add item 6 to the mixture from Step 4, mix well and cool to roomtemperature.

6. Add item I to the mixture from Step 5 and dispense well until auniform suspension is obtained.

We claim:
 1. A method of inducing inhibition of cell proliferation in asolid breast tumor comprising administering to a host in need of saidtreatment from about 10 mg to about 20 mg of trans retinoic acid andfrom about 0.00025 mg to about 0.10 mg of a Vitamin D₃ analog selectedfrom the group consisting of1α,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol,1α,25-dihydroxy-26,27-hexafluorocholecalciferol and1α,25-dihydroxy-16-ene-23-yne-cholecalciferol.
 2. A method in accordancewith claim 1 wherein the Vitamin D₃ analog is1α,25-dihydroxy-26,27-hexafluoro-16-ene-23-yne-cholecalciferol.